A diffusion based long-range and steady chemical gradient generator on a microfluidic device for studying bacterial chemotaxis

Abstract

Studies on chemotaxis in microfluidics device have become a major area of research to generate physiologically similar environment in vitro. In this work, a novel micro-fluidic device has been developed to study chemo-taxis of cells in near physiological condition which can create controllable, steady and long-range chemical gradients using various chemoeffectors in a micro-channel. Hydrogels like agarose, collagen, etc, can be used in the device to maintain exclusive diffusive flux of various chemical species into the micro-channel under study. Variations of concentrations and flow rates of Texas Red dextran in the device revealed that an increase in the concentration of the dye in the feed from 6 to 18 μg ml −1 , causes a steeper chemical gradient in the device, whereas the flow rate of the dye has practically no effect on the chemical gradient in the device. This observation confirms that a diffusion controlled chemical gradient is generated in the micro-channel. Chemo-taxis of E. coli cells were studied under the steady gradient of a chemo-attractant and a chemo-repellent separately in the same chemical gradient generator. For sorbitol and NiSO 4 •6H 2 O, the bacterial cells exhibit a steady distribution in the micro channel after 1 h and 30 min, respectively. From the distribution of bacterial population chemo-tactic strength of the chemo-effectors was estimated for E. coli. In a long microfluidic channel, migration behavior of bacterial cells under diffusion controlled chemical gradient showed chemotaxis, random movement, aggregation, and concentration dependent reverse chemotaxis.

A hybrid microfluidic device for on-demand orientation and multidirectional imaging of C. elegans organs and neurons

“…Recent development of non-flowing gradient generators include static gradients generated by diffusion of molecules through porous nitrocellulose 7 or polyester 38 membranes, agarose hydrogel, 15,39,40 collagen, 10,41 polyethylene glycol (PEG) hydrogels, 42,43 through micro-jet array perfusion channels with minimal flow, 44,45 or through in situ biofabricated biopolymer membranes. 46,47 By restricting convective flow with membranes or hydrogels while allowing diffusion of small molecules to generate chemical gradients, flow-free and diffusion-based static gradient generators are able to decouple cell motion of nonadherent cells from flow.…”

“…These problems suggest that a static, non-flowing gradient generator may be a more suitable platform for chemotaxis studies in the long run, despite posing greater difficulties in design, fabrication, and packaging. 7,12,[14][15][16] …”

“…Rusconi et al calculated a "chemotactic index" for bacteria in a gradient of oxygen (also a chemoattractant), and demonstrated a significant drop-off in this index as the shear rate increased. It is intriguing to note that this shear trapping effect depletes bacterial cells from the center of a microchannel, while the finding by Locsei and Pedley 15 hints that chemotaxis is effective only in the center of a microchannel where shear is below the threshold of 2 s…”

Interplay of chemical and thermal gradient on bacterial migration in a diffusive microfluidic device

“…As chemical and thermal gradients are among the most important agents affecting the migration pattern of microbial cells within a physiological system, the effects of these individual gradients on E. coli have been reported by researchers. 5,[8][9][10][11][12][13][14][15] Earlier, conventional methods such as capillary assay 8 and Boyden chamber 9 have been used to generate chemical gradients to study the chemotaxis of E. coli cells in vitro. Agar plates placed over aluminum blocks with provision for the flow of hot and cold fluids 10 were used to generate thermal gradients to study the thermotaxis of E. coli cells.…”

“…5 This is due to the fact that microfluidic environments can successfully mimic the requisite physiological conditions, both qualitatively and quantitatively. There have been reports on individual studies of E. coli cell migration due to chemical 5,[11][12][13] and thermal gradients 14,15 using microfluidic devices.…”